This FIRCA application is based on the parent grant 2-R37-CA40333 "Human multidrug resistance (P-glycoprotein) genes" (MERIT Award). The main goal of the parent grant is to study a clinically important mechanism of multidrug resistance in tumor cells, which is mediated by P-glycoprotein, an ATP-dependent efflux pump for multiple hydrophobic compounds. A major part of this study involves the identification and characterization of mutations in the MDR1 (P-glycoprotein)gene that alter the substrate specificity of P-glycoprotein-mediated drug transport. Analysis of these mutants helps in understanding the mechanism of P-glycoprotein action, designing novel P-glycoprotein inhibitors and developing "drug-tailored" variants of the MDR1 gene for bone marrow chemoprotection. A new mechanistic approach to the analysis of altered-specificity MDR1 mutants has been developed by our foreign collaborator (FC). In this approach, different forms of P-glycoprotein are expressed in Sf9 insect cells using a baculoviral vector. P-glycoprotein in the isolated Sf9 membrane preparations possesses an ATPase activity, which is strongly and specifically stimulated by P-glycoprotein-transported compounds. The FC has demonstrated that a mutant form of P-glycoprotein, analyzed in this system, shows an altered profile of ATPase stimulation by different drugs. These changes parallel the changes in the profile of cross-resistance to different drugs conferred by the same mutant in mammalian cells. A comparison of the profiles of ATPase simulation and cross-resistance for other altered-specificity MDR1 mutants should provide important mechanistic information. The PI's laboratory is using a retroviral mutation-selection system to identify novel altered-specificity MDR1 mutations. The mutant MDR1 cDNA sequences will be provided to the FC, who will express the mutant P-glycoproteins in Sf9 cell membranes and analyze the ability of different drugs and competitive inhibitors of P- glycoprotein to stimulate the ATPase activity of these mutants. These data will be correlated with the drug-resistance and binding properties of the same mutants, analyzed in the PI's laboratory. The results of this study will be used to identify the determinants of the P-glycoprotein substrate specificity and the relationship between the drug transport and ATPase activities of P-glycoprotein.